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The effect of polyol compounds on the thermostability of penicillin G acylase from a mutant of Escherichia coli ATCC 11105
ProcessBiochemiwy,Vol. 30,No. 2, pp. 133-139, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights resewed 0032-9592/95 $9.50 + 0.00 ELSEVIER
The Effect of Polyol Compounds on the Thermostabilityof PenicillinG Acylase from a Mutant of Escherichiu coli ATCC 11105 Altan Erarslan The Scientific and Technical Research Council of Turkey, Marmara Research Centre, Institute of Genetic Engineering and Biotechnology, PO Box 2 1,4 1470 Gebze, Kocaeli, Turkey (Received 22 December 1993; accepted 12 March 1994)
The e#ects of the I5polyol compounds on the thermostabihty ofpenicillin G acylase (PGA) from a mutant of Escherichia coli ATCC 11105 were investigated. PGA solutions containing glucose, sucrose, mannose, adonitol, sorbitol or xylitol at I M concentration showed an initial activity rate (IAR) higher than 3. The IAR values of these six sugars at 50°C increased with their concentrations in PGA solution. The investigations on half-life times and stabilization factor (SF) values of PGA at 45 and SOY, in the presence of these compounds, showed a tendency for the half-life and SF of the enzyme, at SOY, to increase with the OH/C ratios of polyol compounds. Sucrose and glucose were the only two compounds having higher SF values at 50°C than at 45°C. Sucrose showed almost twice the half-life of giucose and was seiected as the best s&biker of PGA activity against thermal inactivation. PGA showed almost the samepH and temperature activity profiles in both presence and absence of sucrose. K, and V, values decreased with the increasing sucrose concentrations, with a greater decrease in K, values.
cular cross-linking using bifunctional reagents, or by the modification of key groups in the tertiary structure of proteins, with reactions such as acylation and reductive alkylation, (iii) protein engineering and (iv) addition of stabilising agents including neutral salts, chelating agents, albumins and other proteins, and thiol reducing agents.le3 Another possible approach to increasing the stability of soluble enzymes is to use additives such as sugar polyols that preserve the catalyst activity by modulating its micro environment. The addition of these polyols prevents the unfolding of protein by strengthening hydrogen bonds or hydrophilic interactions. The efficiency of stabilisation is related to the chemical character of the
INTRODUCTION The rapid inactivation of enzymes at elevated temperatures is a major constraint in their biotechnological applications. Enzymes should be stable for long time periods, under operational conditions, in order to be suitable for technological applications. The lengthening of operational stability will reduce the number of enzyme replacements and therefore decrease the overall cost of enzyme use. Four methods can be considered for the stabihsation of enzymes: (i) screening of micro-organisms for enzymes with enhanced intrinsic stability, (ii) chemical modihcation of enzyme molecules, either by intramole133
134
Altan Erarslan
additive and the enzyme molecules. Enzymes stabilised by this method can be applied conveniently in industrial processes involving non-Newtonian reaction media.4J Little work related to stabilisation of PGA by the addition of polyol compounds into enzyme solutions has appeared in the literature. We have previously reported on the activity and stability of penicillin G acylase (PGA) from a mutant strain of E. cob ATCC 11 105.7-9 In this paper, the effect of polyol compounds on the thermostability of PGA obtained from the same strain is reported. MATERIALS AND METHODS Chemicals Penicillin G (Pen G) and 6-aminopenicillanic acid (6-APA) were kindly provided by Unifar Chemical Ltd, Istanbul, Turkey. DEAE cellulose used for enzyme purification was obtained from Sigma Chem. Ltd, USA. Polyol compounds and all other chemicals used were of analytical grade and supplied either by Merck AG, Germany or Sigma Chem. Ltd, USA. Microorganism A mutant of Escherichia coli ATCC 11105 was obtained by chemical mutagenesis with N-methylN’-nitro-N-nitroso-guanidine (NTG) as described elsewherelO and used throughout this work. A simple microbiological Serratia marcencens overlay technique was used to differentiate mutant colonies from the parent strain. This method is based on the use of S. rnarcencens, which is sensitive to 6-APA but resistant to Pen G. Penicillin G acylase producing mutants were screened by comparing their inhibition zone diameters with that of the wild type PGA producer E. coli ATCC 11105. The mutant strain is deposited in the culture collection of our department, Media and culture conditions The production of PGA by cultivation of E. coli in a jar fermenter (Biostat E, Braun Melsungen GmbH, Germany) was carried out under the same medium and culture conditions as described previously. ” The fermentation temperature, pH, and dissolved oxygen concentration were 28°C 7.0 and 10% of saturation, respectively. PGA synthesis was induced after 6 h of incubation by the continuous feeding of phenyl acetic acid
(PAA) into the fermenter of 0.3% (w/v).
to a final concentration
Enzyme purification Intracellular PGA was extracted from the mutant strain after cell disruption and purified by DEAEcellulose ion-exchange chromatography followed by preliminary precipitation steps as described previously.10~‘2 Determination of enzyme activity The hydroxyl amine method of Batchelor et aLI was used to determine enzyme activity during purification. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of 6-APA min-’ at 40°C and pH 8.0 from 15 mM Pen G in 50 rmr phosphate buffer. Since this method is not sensitive enough for kinetic investigations, the p-dimethylaminobenzaldehyde (PDAB) method was used.14 In this method, one unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of 6-APA mir- ’from 15 ells Pen G at 40°C in 50 II~Mphosphate buffer at various pH values. Protein measurement Protein was measured by Coomassie Blue binding using bovine serum albumin (BSA) as the standard.15,16 Estimation of deactivation rate constants of PGA Deactivation rate constants ( kd) of PGA at different temperatures, in media containing various individual polyol compounds, were estimated according to theoretical considerations described previously.’ A O-05 ml sample of PGA solution (specific activity; 2 1.250 U mg- ‘, protein concentration; 0.48 mg ml-‘) was mixed with 0.45 ml of 50 mM phosphate buffer (pH, 8.0) containing polyol compounds and incubated for different time intervals at various temperatures. Thereafter 0.5 ml 30 mM Pen G in 50 mM phosphate buffer (pH, 8.0) was added to the mixture and incubated for 3 min at 40°C. Activity was measured by the PDAB method and k, values were calculated from the slope of ln( E,/E,)= - k,t (Ei; initial activity of inactivated form of PGA, E,; activity of initial or transient form of PGA, t; time). The expression of the influence of polyol compounds The influence of polyol compounds on the thermal inactivation of PGA was expressed as
Thermostability of penicillin G acylase
initial activity rate constants (IAR) and calculated by the following formula: lAR = (initial activity)po,yo,_PGAmix/ (initial activity),,,i,,
PGA
The expression of the thermal stability of PGA in polyol compounds The thermal stability of PGA in polyol compounds was expressed as a stabilisation factor (SF) and calculated by the following formula: SF = (half-life time),,,,_pcA
mix/
(half-life time)native PGA Half-life time is the time required for 50% inactivation of enzyme activity and is calculated from the ln(E,/E,) = - kdt equation by placing E,= 2Ei. RESULTS AND DISCUSSION Influence of polyol compounds on PGA The influence of 15 polyol compounds, including disaccharides (sucrose and trehalose; OH/ C = 152) hexoses (glucose, sorbose, mannose and galactose; OH/C = 1.20: mannitol, sorbitol and inositol; OH/C = 1.00: rhamnose; OH/C = 152) pentoses (adonitol and xylitol; OH/C = 1.0: arabinose; OH/C = l-25) tetroses (erythritol; OH/ C = 1.00) and trioses (glycerol; OH/C = l-00) were investigated on the deactivation of PGA at 50°C after 30 min incubation of enzyme. Effects on deactivation were expressed as IAR values (Fig. 1). With the exception of glycerol, all of the polyol compounds enhanced the IAR value and consequently the initial activity of PGA. Enhancement was not, however, dependent on the OH/C ratio of polyol compounds.
135
The polyol compounds which provide an IAR value higher than three (sucrose, glucose, sorbitol, mannose, adonitol and xylitol) were selected in order to make further investigations on the thermostability of PGA. The effect of the concentration of these six polyols on the initial reaction rate of PGA at 50°C is shown in Fig. 2. Decreases were observed on the initial activity of enzyme with decreases of polyol concentration. This indicates that the protective effect of polyols on PGA activity at 50°C decreased at concentrations below 1 M. Thermal deactivation kinetics of PGA in the presence of polyol compounds In our previous work, we reported the inactivation of PGA according to a mechanism of first order thermal deactivation kinetics upon prolonged exposure to heat.’ Deactivation kinetics of PGA at 45 and 50°C were re-investigated in this work, in order to estimate the apparent deactivation rate constants ( kd) of enzyme in the presence of each of six polyol compounds. The k, values were found from the slope of linear regression lines, obtained by plotting ln( E,/E,) values vs time (Figs 3 and 4) and used in the calculation of the half-life time. Half-life times and SF values of PGA at 45 and 50°C in the presence of these compounds are summarized in Table 1. The half-life and SF values of enzyme were found to vary over a large scale, particularly, at 45°C and the OH/C ratio appeared to have no effect on the stabilising factor of polyol on PGA. However, an increase was observed (at 50°C) of the half-life and SF values of enzyme with the increase of OH/C ratios of polyol compounds.
I Glucose
_
“x$iE
_
0 0
1
2
3
4
IAR value
Fig. 1. Influence of 15 polyols on the initial reaction rate of PGA at 50°C after 30 min incubation of enzyme.
0.1
0.2
0.3
0.4
0,5
0.6
Polyol conccllbation,
0.7
0,8
0.9
I
(hq
Fig. 2. The effect of the concentration of polyols having an IAR value higher than 3 on the initial reaction rate of PGA at 50°C after 30 min incubation of enzyme.
Altan Era&an
136
0
s-
o
Gluasc
A
sorbit
OMramnc
-0.7
0
A&nib4
v
Xyliti
M 0
30
60
90
120
150
180
Time, @in)
lines obtained by linear regression for the Fig. 3. Best-fit thermal deactivation of PGA at 45°C in the presence of polyol compounds having an IAR value higher than 3. Equations of best-fit lines and r values are as follows: sucrose, ln( E,/E,)\= (1~89250f1~17397)\~10-*+I( -1.72393 k gh~cose, ln(E,/E,) 0-108534)x 1O-3 t, r= -0.990236; =(2.62200+3,72362)x 10-2+(3.52233+0.337814)x 1O-3 t, r= -0.990927; sorbitol, Ln(E,/E,)= (0.77619 + 1.00799) x lo-*+( - 1.91438+@110976)~ 1O-3 t, r= -@993346; ln(E,/E,)=(1.44000+ 1.04475)x lo-*+ mannose, ( - 2.80300 + 0.142172) x 10m3 t, r= - 0.996163; adonitol, In (,5,/E,) = ( - 2.35905 f 1.90233) x lo- 2 + - 4.48390 k xylitol, In (El/E,)= 0.209439)x lo-” t, r= -0.995665; (4.11810+ 2~38198)~10-~+( -3.76819+0*262247)x 10e3t, r= -0.990452. The apparent deactivation rate constant (kd, mix- ‘) of PGA containing each of these polyols was calculated from the slope of these lines.
0
0
s-
-0.4
0
Gluuac
-0.6
A
Sotitol
0
Maws
42
3
-1
-0.8
-1.4 -1.6
0
Adonitol
v
Xylitol
-1,s -2 -2.2 0
30
60
PO
120
150
11m. (min)
Fig. 4. Best-fit lines obtained by linear regression for the thermal deactivation of PGA at 5O’C in the presence of polyol compounds having an IAR value higher than 3. Equations of best-fit lines and r values are as follows: sucrose, In(E,/E,)=(-1~55762+1*08518) ~10-~+( -8.7501Ok 0,119474) x 10m3 t, r= - O-999627; glucose, ln( EJE,) = ( -0.92000 + 1.17052) x lo-~*+( - 1.65317 k 0.01593) x 10m2 t, r= - 0.999861; sorbitol, In{ EJE,) = ( -0-30000 f 3.19368) x lo-*+ ( -2.31800 * 0.08692) x 1O-2 t, r= -0.997897; mannose, ln( E,/E,) = ( - 1.32000 f 2.96837) x 10-2+ (-2*60630+0*121183) x 10-2t, r= -0336773 r= - 0.996773. adonitol h&Y/E,) = ( - 4.73524 + :.93288)x 10-2+()-2.81526f)O-0968?)x lO-2 - 0.99764; xylitol, ln(EJE,)= ( - 1.06625 f 2.26750;‘~ l(? + ( - 3.29648 + 0-067604)x 1O-2 t, r= - 0.99916. The apparent deactivation rate constant (kdr mix-‘) of PGA containingeach of these polyols was calculated from the slope of these lines.
Table 1. Half-life time and stabiiation factor (SF) for penicillin G acylase stored in buffer and in the presence of various polyol compounds Polyol compound
OH/C ratio
Half-life (min) 45°C
Sucrose Glucose Sorbitol Mannose Adonitol XyIitol Buffer
1.52 1.20 l-00 1.20 l-00 l-00 -
402.00 196.74 362.00 247.24 154-55 183.91 126.00
Stabiliration factor (SF)
50°C
79.20 41.93 29.30 26.60 24.62 21.11 2000
45°C
50%.
3.19 le.56 2.87 1.96 1-22 1.46 1.00
3.96 2.10 1.47 1.33 1,23 1.05 1.00
We have not found any report in the literature relating to the effects of polyol compounds on the thermostability of PGA from E. cd, or other PGA producing strains, in order to make comparisons and discussions with our results. On the other hand, two epimer polyols, xylitol and adonitol had an influence on enzyme thermostability in the same range of concentrations. Larreta-Garde et aL5 reported stabilisation by xylitol, but destabilisation by adonitol, of the activity of yeast alcohol dehydrogenase. Some epimer polyols, which differ just by one hydroxyl position, can show opposite behaviour towards the stabilisation of an oxidoreductase enzyme such as yeast alcohol dehydrogenase, however, such behaviour was not observed on a hydrolytic enzyme PGA. Sucrose and glucose were the two compounds having higher stabilisation factor (SF) values at 50°C than at 45°C. This indicates that the protective effects of these sugars on PGA activity were better than that of the other four polyols. Sucrose showed almost two-fold higher half-life and SF values than glucose and consequently was selected as the best stabiliser of PGA activity against thermal inactivation. Further kinetic investigations were made only with sucrose. Temperature and pH profiles of PGA in the presence of sucrose The relationship of temperature, and the activities of PGA in the absence and presence of sucrose ( 1~) was determined by measuring the initial reaction rate at pH 8.0 and at temperatures between 35 and 70°C. The optimum temperature for Pen G hydrolysis by PGA in both cases was 60°C. Relative activities at other temperatures were estimated, considering the activity at 60°C as 100% (Fig. 5).
Thennostability of penicillin G acylase
30
40
50 Tem&mh=e.
60
6
70
7
Fig. 5. Temperature-activity profile of PGA from a mutant of E. coli ATCC 11105 in the absence and oresence of 1 M sucrose. In the case of sugar-free enzyme (buffer) O-05 ml PGA (specific activity; 2 1.25 U mg - I, protein concentration; 0.48 mg ml-‘) was mixed with 0.95 ml 15 mu Pen G solution in 50 mu phosphate buffer, pH 8.0, and incubated for 3 min at various temperatures. In the case of sucrose containing enzyme, 0.05 ml PGA was mired with 0.45 ml 2 M sucrose and @5 ml 30 mM Wn G solutions prepared in 50 mu phosphate buffer, pH S-0, and incubated at various temperatures. Activity was measured by the PDAB method.
9
8 PH
W)
6. pH-activity profile of PGA from a mutant of E. coli ATCC 11105 in the absence and presence of 1 M sucrose. In the case of sugar-free enzyme (buffer) 0.05 ml PGA (specific activity; 21.25 U mg-‘, protein concentration; 0.48 mg ml- ‘) was mixed with @95 ml 15 mu Pen G solution prepared in 50 mu phosphate buffer in various pH values and incubated at 40°C for 3 min. In the case of sucrose-containing enzyme, 0.05 ml PGA was mixed with @45 ml 2 Msucrose and 0.5 ml 30 mu Pen G solutions prepared in 50 mu phosphate buffers at various pH values and incubated at 40°C for 3 min. Activity was measured by the PDAB method.
Fii.
0.15
The effects of pH on PGA activity were determined by measuring the initial reaction rates at 40°C and at pH values between 6.0 and 9-O The pH activity profiles are shown in Fig. 6. The highest initial reaction rates were obtained at pH 7.5 with 1 M sucrose-containing PGA, and at pH 8.0 with sucrose-free PGA. The initial rates at other pH values were expressed as relative reaction rates, considering the values at pH 7.5 and 8-O as 100% for sucrose-containing and sucrose-free PGA, respectively. The presence of sucrose provided an appropriate pH range between 7.5 and 8.5 for Pen G hydrolysis, whereas sugar-free PGA showed a single optimal pH value at pH 8-O. The effect of sucrose on V, and AK,,, values of PGA In the absence and presence of sucrose, V, and K,,, values of PGA were obtained by an enzyme preparate having 21.25 U mg-’ specific activity and 0.48 mg ml-l protein concentration. Reactions for activity measurements were carried out for 3 mm at 40°C in 50 mu phosphate buffer, pH 8.0. The V,,,and K, values of enzyme in the presence of 0.5 M and 1 M sucrose were estimated from a Lineweaver-Burk plot (Fig. 7) as 15*36+0.57 U ml-’ mint and 0.88+0*10 mu Pen G, and 14.32f0.09 U ml-’ min-’ and @79 f 0.02 mu Pen G, respectively. The V, and K, values of PGA were estimated from the same plot as 20.84 & 0.03 U ml-’ rnin-’ and
0
0.13 0.12 0.11 -r
0.5MSucmse
L-J
0.14
0.1 0.09
0
l.OMSuCmse
A
o.o~su~mae
0.08 0.07 0.06 0.05 0.04 0.03
0
0.2
0.4
0.6
0.8
1
WI Fig. 7. Lineweaver-Burk plot of PGA from a mutant of E. coli ATCC 11105 in the absence and presence of @5 Mand 1 M sucrose, to determine the K, and V,, values. Equations of best-fit lines, obtained by linear regression and r ialues, are as follows: 0 M sucrose. 1/~=(4.79861+0.23614)X lo-‘+ (1.27984 + @05902) x 10-l 11/S), r= -0.996825; @5 lo-*+(5.70752+ M sucrose, l/v =(6*51061 f0.23948)~ 0.42262)x lo-* (l/S), r= -0.994562; 1 M sucrose, l/v= (7~08070f004693)\x)10-‘~+~(5~56148rtO=10181)~x 1O-2 (l/S), r= - 0.999671.
2.67 f 0.02 ~-IMPen G, respectively, in the absence of sucrose. The comparison of V, and K, values in sucrose-containing PGA to those of sucrose-free PGA in buffer solution ( If,,,, and K,,,J showed that K, and V, values decreased with increasing concentrations of added sucrose, but the percentage of decrease was higher in Km values (Fig. 8). This can be explained by an increase of diffusional resistance to the Pen G in concentrated sucrose media. Results suggest that the more the
Altan Erarsh
138
was higher in sucrose-containing media up to 5 mu concentrations of Pen G (Fig. 7). Consequently the activation energy of sucrose-containing enzyme decreased from 10-l 5 + O-43 to 8.17 k O-58 kcal mol- ‘. This indicates that the presence of sucrose contributes to a decrease in the activation energy of PGA.
loo 80
0
3 6o E
L
40
of
I.,
0
8,
0.2
0.4
I.
I.
0.6
0,8
/
lo
I
SucmwCnnccotmtion.(M)
Fig. 8. Effect of sucrose concentration on V, and K, values of PGA from a mutant of E. coli ATCC 11105 for Pen G hydrolysis.
ACKNOWLEDGEMENTS This work was supported by funds from the NATO-SFS Program (Tu-842 Biotech). The author wishes to kindly acknowledge the contributions of Halil Kocer, a biologist in our Institute, for his technical assistance.
3.6 3.4
REFERENCES
3.2 3 2.8 ' 9
2.6 2.4 2.2 2 1,s
I.6
F., 3
,. 3.05
, , , . , , , , , , , , , , , , .y 3.1
3.15
3.2
3.25
3.3
3.35
[lrr'1*lo".(Kr'
plot for the estimation of activation Fig. 9. Arrhenius energies of PGA from a mutant of E. coli ATCC 11105 in the absence and presence of 1 M sucrose. Equations of bestfit lines obtained by linear regression and r values are as In v=(1~87816f0~0695197)~ lOI+ follows: buffer, (-5.10879f0.217429)~ 10m3 (l/T), r= -@995502; sucrose, In v=(1~56448~0~0914875)x lOl+(-4.10958+ 0.293123) X 10m3 (l/T), r= - 0.989978.
water structure of enzyme was affected by increased sucrose concentration, the more the initial activity of enzyme decreased. Water is a reactant of hydrolytic reactions and, as sucrose tends to make a fraction of the water unavailable, the water activity and thus the enzyme activity are reduced. Effect of sucrose on the activation energy of PGA The activation energy of PGA in the absence and presence of 1 M sucrose was calculated from the slope of the Arrhenius plot (Fig. 9) as 10.15 t-O-43 and 8.17 + 0.58 kcal mol-‘, respectively. In general, the activation energy of a reaction is expected to be increased when a decrease in the reaction rate occurs. Within the temperature ranges tested from 35°C to 70°C the Arrhenius plot of Pen G hydrolysis was close to first order. The initial reaction rate of Pen G hydrolysis
1. Schmid, R. D., Stabilized soluble enzymes. A& Biothem. Eng., 12 (1979) 41-127. of enzymes against 2. Klibanov,- A. M., Stabilization thermal inactivation. Adv. Au.& _. Microbial.. 29 i 1983) l-28. 3. Janecek, s., Strategies for obtaining stable enzymes. Proc. B&hem.. 28 (1993) 435-45. ‘Xu, 2. F., Biton, J. & Thomas, D., 4. Larreta-Garde,‘V., Stability of enzymes in low water activity media. Biocatalysis in Organic Media, ed. J. Tramper & M. D. Lilly Proc. Znt. Symp., Wageningen, The Netherlands, 1986, pp. 247-52. 5. Larreta-Garde, V., Xu, Z. F. & Thomas, D., Behavior of enzymes in the presence of additives. Enzyme Engineerina. ed. H. W. Blanch & A. M. Klibanov. Vol. 9. 1988. ~6’294-8. 6. Erarslan, A., The effect of pH on the kinetics of penicillin G acylase obtained from a mutant of Escherichiu coli ATCC 11105. Proc. Biochem., 28 (1993) 319-24. 7. Erarslan, A. & Kocer, H., Thermal inactivation kinetics of penicillin G acyiase obtained from a mutant derivative of Escherichiu coli ATCC 11105. J. Chem. Technol. Biotechnol., 55 (1992) 79-84. of 8. Erarslan, A. & Giiray, A., Kinetic investigation penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105 immobilized on oxirane-acrylic beads. J. Chem. Technof. Biorechnol., 5 1 ( 1991) 181-95. 9. Erarslan, A., Dagagan, L., Giiray, A., Terzi, 1. & Bermek, E., Several immobilization methods of penicillin acylase purified from Escherichia coli ATCC 11105 and kinetic properties of enzyme immobilized on carboxy methyl cellulose. Doga-Tr. J.. Eng. Env. Sci., 14 (1990) 381-8. 10. Era&m, A., Terzi, I., Giiray, A. & Bermek, E., Purification and kinetics of penicillin G acylase from a mutant strain of Escherichiu coli ATCC 11105. J. Chem. Technol. Biotechnol., Sl(l991) 27-40. 11. Erarslan, A. & Giiray, A., Fermentation of penicillin G acylase by a mutant strain of Escherichia co/i ATCC lllOS.Dojja-Tr.L Biology, 15(1991) 167-74. 12. Erarslan, A., Dagavan, L., Giiray, A., Terzi, i. & Bermek, E., Purification and kinetic properties of penicillin acylase from Escherichia coli ATCC 11105. Doga-Tr. J. Biology, 13 (1989) 8-13.
Themstability
of penicillin G acylase
13. Batchelor, F. R., Chain, E. B., Hardy, T. L., Mansford, K.
R L. & Rolinson, G. N., 6-Aminopenicillanic acid. III. Isolation and purification. Proc. Roy. Sot., B, 154 (1961) 498-508. 14. Shewale, G. J., Kumar, K. K. & Ambekar, G. R., Evaluation and determination of 6-aminopenicillanic acid by pdimethylaminobenzaldehyde. Biotechnol. Techniques, 1 (1987) 69-72.
139
15. Spector, T., Refinement of the Coomassie Blue method of protein quantitation. Anal. Biochem., 86 (1978) 142-6. 16. Sedmak, J. J. & Grossberg, S. E., A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G-250. Anal. Biochem., 79 (1977) 544-52.
The Effect of Polyol Compounds on the Thermostabilityof PenicillinG Acylase from a Mutant of Escherichiu coli ATCC 11105 Altan Erarslan The Scientific and Technical Research Council of Turkey, Marmara Research Centre, Institute of Genetic Engineering and Biotechnology, PO Box 2 1,4 1470 Gebze, Kocaeli, Turkey (Received 22 December 1993; accepted 12 March 1994)
The e#ects of the I5polyol compounds on the thermostabihty ofpenicillin G acylase (PGA) from a mutant of Escherichia coli ATCC 11105 were investigated. PGA solutions containing glucose, sucrose, mannose, adonitol, sorbitol or xylitol at I M concentration showed an initial activity rate (IAR) higher than 3. The IAR values of these six sugars at 50°C increased with their concentrations in PGA solution. The investigations on half-life times and stabilization factor (SF) values of PGA at 45 and SOY, in the presence of these compounds, showed a tendency for the half-life and SF of the enzyme, at SOY, to increase with the OH/C ratios of polyol compounds. Sucrose and glucose were the only two compounds having higher SF values at 50°C than at 45°C. Sucrose showed almost twice the half-life of giucose and was seiected as the best s&biker of PGA activity against thermal inactivation. PGA showed almost the samepH and temperature activity profiles in both presence and absence of sucrose. K, and V, values decreased with the increasing sucrose concentrations, with a greater decrease in K, values.
cular cross-linking using bifunctional reagents, or by the modification of key groups in the tertiary structure of proteins, with reactions such as acylation and reductive alkylation, (iii) protein engineering and (iv) addition of stabilising agents including neutral salts, chelating agents, albumins and other proteins, and thiol reducing agents.le3 Another possible approach to increasing the stability of soluble enzymes is to use additives such as sugar polyols that preserve the catalyst activity by modulating its micro environment. The addition of these polyols prevents the unfolding of protein by strengthening hydrogen bonds or hydrophilic interactions. The efficiency of stabilisation is related to the chemical character of the
INTRODUCTION The rapid inactivation of enzymes at elevated temperatures is a major constraint in their biotechnological applications. Enzymes should be stable for long time periods, under operational conditions, in order to be suitable for technological applications. The lengthening of operational stability will reduce the number of enzyme replacements and therefore decrease the overall cost of enzyme use. Four methods can be considered for the stabihsation of enzymes: (i) screening of micro-organisms for enzymes with enhanced intrinsic stability, (ii) chemical modihcation of enzyme molecules, either by intramole133
134
Altan Erarslan
additive and the enzyme molecules. Enzymes stabilised by this method can be applied conveniently in industrial processes involving non-Newtonian reaction media.4J Little work related to stabilisation of PGA by the addition of polyol compounds into enzyme solutions has appeared in the literature. We have previously reported on the activity and stability of penicillin G acylase (PGA) from a mutant strain of E. cob ATCC 11 105.7-9 In this paper, the effect of polyol compounds on the thermostability of PGA obtained from the same strain is reported. MATERIALS AND METHODS Chemicals Penicillin G (Pen G) and 6-aminopenicillanic acid (6-APA) were kindly provided by Unifar Chemical Ltd, Istanbul, Turkey. DEAE cellulose used for enzyme purification was obtained from Sigma Chem. Ltd, USA. Polyol compounds and all other chemicals used were of analytical grade and supplied either by Merck AG, Germany or Sigma Chem. Ltd, USA. Microorganism A mutant of Escherichia coli ATCC 11105 was obtained by chemical mutagenesis with N-methylN’-nitro-N-nitroso-guanidine (NTG) as described elsewherelO and used throughout this work. A simple microbiological Serratia marcencens overlay technique was used to differentiate mutant colonies from the parent strain. This method is based on the use of S. rnarcencens, which is sensitive to 6-APA but resistant to Pen G. Penicillin G acylase producing mutants were screened by comparing their inhibition zone diameters with that of the wild type PGA producer E. coli ATCC 11105. The mutant strain is deposited in the culture collection of our department, Media and culture conditions The production of PGA by cultivation of E. coli in a jar fermenter (Biostat E, Braun Melsungen GmbH, Germany) was carried out under the same medium and culture conditions as described previously. ” The fermentation temperature, pH, and dissolved oxygen concentration were 28°C 7.0 and 10% of saturation, respectively. PGA synthesis was induced after 6 h of incubation by the continuous feeding of phenyl acetic acid
(PAA) into the fermenter of 0.3% (w/v).
to a final concentration
Enzyme purification Intracellular PGA was extracted from the mutant strain after cell disruption and purified by DEAEcellulose ion-exchange chromatography followed by preliminary precipitation steps as described previously.10~‘2 Determination of enzyme activity The hydroxyl amine method of Batchelor et aLI was used to determine enzyme activity during purification. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of 6-APA min-’ at 40°C and pH 8.0 from 15 mM Pen G in 50 rmr phosphate buffer. Since this method is not sensitive enough for kinetic investigations, the p-dimethylaminobenzaldehyde (PDAB) method was used.14 In this method, one unit of enzyme activity is defined as the amount of enzyme required to produce 1 pmol of 6-APA mir- ’from 15 ells Pen G at 40°C in 50 II~Mphosphate buffer at various pH values. Protein measurement Protein was measured by Coomassie Blue binding using bovine serum albumin (BSA) as the standard.15,16 Estimation of deactivation rate constants of PGA Deactivation rate constants ( kd) of PGA at different temperatures, in media containing various individual polyol compounds, were estimated according to theoretical considerations described previously.’ A O-05 ml sample of PGA solution (specific activity; 2 1.250 U mg- ‘, protein concentration; 0.48 mg ml-‘) was mixed with 0.45 ml of 50 mM phosphate buffer (pH, 8.0) containing polyol compounds and incubated for different time intervals at various temperatures. Thereafter 0.5 ml 30 mM Pen G in 50 mM phosphate buffer (pH, 8.0) was added to the mixture and incubated for 3 min at 40°C. Activity was measured by the PDAB method and k, values were calculated from the slope of ln( E,/E,)= - k,t (Ei; initial activity of inactivated form of PGA, E,; activity of initial or transient form of PGA, t; time). The expression of the influence of polyol compounds The influence of polyol compounds on the thermal inactivation of PGA was expressed as
Thermostability of penicillin G acylase
initial activity rate constants (IAR) and calculated by the following formula: lAR = (initial activity)po,yo,_PGAmix/ (initial activity),,,i,,
PGA
The expression of the thermal stability of PGA in polyol compounds The thermal stability of PGA in polyol compounds was expressed as a stabilisation factor (SF) and calculated by the following formula: SF = (half-life time),,,,_pcA
mix/
(half-life time)native PGA Half-life time is the time required for 50% inactivation of enzyme activity and is calculated from the ln(E,/E,) = - kdt equation by placing E,= 2Ei. RESULTS AND DISCUSSION Influence of polyol compounds on PGA The influence of 15 polyol compounds, including disaccharides (sucrose and trehalose; OH/ C = 152) hexoses (glucose, sorbose, mannose and galactose; OH/C = 1.20: mannitol, sorbitol and inositol; OH/C = 1.00: rhamnose; OH/C = 152) pentoses (adonitol and xylitol; OH/C = 1.0: arabinose; OH/C = l-25) tetroses (erythritol; OH/ C = 1.00) and trioses (glycerol; OH/C = l-00) were investigated on the deactivation of PGA at 50°C after 30 min incubation of enzyme. Effects on deactivation were expressed as IAR values (Fig. 1). With the exception of glycerol, all of the polyol compounds enhanced the IAR value and consequently the initial activity of PGA. Enhancement was not, however, dependent on the OH/C ratio of polyol compounds.
135
The polyol compounds which provide an IAR value higher than three (sucrose, glucose, sorbitol, mannose, adonitol and xylitol) were selected in order to make further investigations on the thermostability of PGA. The effect of the concentration of these six polyols on the initial reaction rate of PGA at 50°C is shown in Fig. 2. Decreases were observed on the initial activity of enzyme with decreases of polyol concentration. This indicates that the protective effect of polyols on PGA activity at 50°C decreased at concentrations below 1 M. Thermal deactivation kinetics of PGA in the presence of polyol compounds In our previous work, we reported the inactivation of PGA according to a mechanism of first order thermal deactivation kinetics upon prolonged exposure to heat.’ Deactivation kinetics of PGA at 45 and 50°C were re-investigated in this work, in order to estimate the apparent deactivation rate constants ( kd) of enzyme in the presence of each of six polyol compounds. The k, values were found from the slope of linear regression lines, obtained by plotting ln( E,/E,) values vs time (Figs 3 and 4) and used in the calculation of the half-life time. Half-life times and SF values of PGA at 45 and 50°C in the presence of these compounds are summarized in Table 1. The half-life and SF values of enzyme were found to vary over a large scale, particularly, at 45°C and the OH/C ratio appeared to have no effect on the stabilising factor of polyol on PGA. However, an increase was observed (at 50°C) of the half-life and SF values of enzyme with the increase of OH/C ratios of polyol compounds.
I Glucose
_
“x$iE
_
0 0
1
2
3
4
IAR value
Fig. 1. Influence of 15 polyols on the initial reaction rate of PGA at 50°C after 30 min incubation of enzyme.
0.1
0.2
0.3
0.4
0,5
0.6
Polyol conccllbation,
0.7
0,8
0.9
I
(hq
Fig. 2. The effect of the concentration of polyols having an IAR value higher than 3 on the initial reaction rate of PGA at 50°C after 30 min incubation of enzyme.
Altan Era&an
136
0
s-
o
Gluasc
A
sorbit
OMramnc
-0.7
0
A&nib4
v
Xyliti
M 0
30
60
90
120
150
180
Time, @in)
lines obtained by linear regression for the Fig. 3. Best-fit thermal deactivation of PGA at 45°C in the presence of polyol compounds having an IAR value higher than 3. Equations of best-fit lines and r values are as follows: sucrose, ln( E,/E,)\= (1~89250f1~17397)\~10-*+I( -1.72393 k gh~cose, ln(E,/E,) 0-108534)x 1O-3 t, r= -0.990236; =(2.62200+3,72362)x 10-2+(3.52233+0.337814)x 1O-3 t, r= -0.990927; sorbitol, Ln(E,/E,)= (0.77619 + 1.00799) x lo-*+( - 1.91438+@110976)~ 1O-3 t, r= -@993346; ln(E,/E,)=(1.44000+ 1.04475)x lo-*+ mannose, ( - 2.80300 + 0.142172) x 10m3 t, r= - 0.996163; adonitol, In (,5,/E,) = ( - 2.35905 f 1.90233) x lo- 2 + - 4.48390 k xylitol, In (El/E,)= 0.209439)x lo-” t, r= -0.995665; (4.11810+ 2~38198)~10-~+( -3.76819+0*262247)x 10e3t, r= -0.990452. The apparent deactivation rate constant (kd, mix- ‘) of PGA containing each of these polyols was calculated from the slope of these lines.
0
0
s-
-0.4
0
Gluuac
-0.6
A
Sotitol
0
Maws
42
3
-1
-0.8
-1.4 -1.6
0
Adonitol
v
Xylitol
-1,s -2 -2.2 0
30
60
PO
120
150
11m. (min)
Fig. 4. Best-fit lines obtained by linear regression for the thermal deactivation of PGA at 5O’C in the presence of polyol compounds having an IAR value higher than 3. Equations of best-fit lines and r values are as follows: sucrose, In(E,/E,)=(-1~55762+1*08518) ~10-~+( -8.7501Ok 0,119474) x 10m3 t, r= - O-999627; glucose, ln( EJE,) = ( -0.92000 + 1.17052) x lo-~*+( - 1.65317 k 0.01593) x 10m2 t, r= - 0.999861; sorbitol, In{ EJE,) = ( -0-30000 f 3.19368) x lo-*+ ( -2.31800 * 0.08692) x 1O-2 t, r= -0.997897; mannose, ln( E,/E,) = ( - 1.32000 f 2.96837) x 10-2+ (-2*60630+0*121183) x 10-2t, r= -0336773 r= - 0.996773. adonitol h&Y/E,) = ( - 4.73524 + :.93288)x 10-2+()-2.81526f)O-0968?)x lO-2 - 0.99764; xylitol, ln(EJE,)= ( - 1.06625 f 2.26750;‘~ l(? + ( - 3.29648 + 0-067604)x 1O-2 t, r= - 0.99916. The apparent deactivation rate constant (kdr mix-‘) of PGA containingeach of these polyols was calculated from the slope of these lines.
Table 1. Half-life time and stabiiation factor (SF) for penicillin G acylase stored in buffer and in the presence of various polyol compounds Polyol compound
OH/C ratio
Half-life (min) 45°C
Sucrose Glucose Sorbitol Mannose Adonitol XyIitol Buffer
1.52 1.20 l-00 1.20 l-00 l-00 -
402.00 196.74 362.00 247.24 154-55 183.91 126.00
Stabiliration factor (SF)
50°C
79.20 41.93 29.30 26.60 24.62 21.11 2000
45°C
50%.
3.19 le.56 2.87 1.96 1-22 1.46 1.00
3.96 2.10 1.47 1.33 1,23 1.05 1.00
We have not found any report in the literature relating to the effects of polyol compounds on the thermostability of PGA from E. cd, or other PGA producing strains, in order to make comparisons and discussions with our results. On the other hand, two epimer polyols, xylitol and adonitol had an influence on enzyme thermostability in the same range of concentrations. Larreta-Garde et aL5 reported stabilisation by xylitol, but destabilisation by adonitol, of the activity of yeast alcohol dehydrogenase. Some epimer polyols, which differ just by one hydroxyl position, can show opposite behaviour towards the stabilisation of an oxidoreductase enzyme such as yeast alcohol dehydrogenase, however, such behaviour was not observed on a hydrolytic enzyme PGA. Sucrose and glucose were the two compounds having higher stabilisation factor (SF) values at 50°C than at 45°C. This indicates that the protective effects of these sugars on PGA activity were better than that of the other four polyols. Sucrose showed almost two-fold higher half-life and SF values than glucose and consequently was selected as the best stabiliser of PGA activity against thermal inactivation. Further kinetic investigations were made only with sucrose. Temperature and pH profiles of PGA in the presence of sucrose The relationship of temperature, and the activities of PGA in the absence and presence of sucrose ( 1~) was determined by measuring the initial reaction rate at pH 8.0 and at temperatures between 35 and 70°C. The optimum temperature for Pen G hydrolysis by PGA in both cases was 60°C. Relative activities at other temperatures were estimated, considering the activity at 60°C as 100% (Fig. 5).
Thennostability of penicillin G acylase
30
40
50 Tem&mh=e.
60
6
70
7
Fig. 5. Temperature-activity profile of PGA from a mutant of E. coli ATCC 11105 in the absence and oresence of 1 M sucrose. In the case of sugar-free enzyme (buffer) O-05 ml PGA (specific activity; 2 1.25 U mg - I, protein concentration; 0.48 mg ml-‘) was mixed with 0.95 ml 15 mu Pen G solution in 50 mu phosphate buffer, pH 8.0, and incubated for 3 min at various temperatures. In the case of sucrose containing enzyme, 0.05 ml PGA was mired with 0.45 ml 2 M sucrose and @5 ml 30 mM Wn G solutions prepared in 50 mu phosphate buffer, pH S-0, and incubated at various temperatures. Activity was measured by the PDAB method.
9
8 PH
W)
6. pH-activity profile of PGA from a mutant of E. coli ATCC 11105 in the absence and presence of 1 M sucrose. In the case of sugar-free enzyme (buffer) 0.05 ml PGA (specific activity; 21.25 U mg-‘, protein concentration; 0.48 mg ml- ‘) was mixed with @95 ml 15 mu Pen G solution prepared in 50 mu phosphate buffer in various pH values and incubated at 40°C for 3 min. In the case of sucrose-containing enzyme, 0.05 ml PGA was mixed with @45 ml 2 Msucrose and 0.5 ml 30 mu Pen G solutions prepared in 50 mu phosphate buffers at various pH values and incubated at 40°C for 3 min. Activity was measured by the PDAB method.
Fii.
0.15
The effects of pH on PGA activity were determined by measuring the initial reaction rates at 40°C and at pH values between 6.0 and 9-O The pH activity profiles are shown in Fig. 6. The highest initial reaction rates were obtained at pH 7.5 with 1 M sucrose-containing PGA, and at pH 8.0 with sucrose-free PGA. The initial rates at other pH values were expressed as relative reaction rates, considering the values at pH 7.5 and 8-O as 100% for sucrose-containing and sucrose-free PGA, respectively. The presence of sucrose provided an appropriate pH range between 7.5 and 8.5 for Pen G hydrolysis, whereas sugar-free PGA showed a single optimal pH value at pH 8-O. The effect of sucrose on V, and AK,,, values of PGA In the absence and presence of sucrose, V, and K,,, values of PGA were obtained by an enzyme preparate having 21.25 U mg-’ specific activity and 0.48 mg ml-l protein concentration. Reactions for activity measurements were carried out for 3 mm at 40°C in 50 mu phosphate buffer, pH 8.0. The V,,,and K, values of enzyme in the presence of 0.5 M and 1 M sucrose were estimated from a Lineweaver-Burk plot (Fig. 7) as 15*36+0.57 U ml-’ mint and 0.88+0*10 mu Pen G, and 14.32f0.09 U ml-’ min-’ and @79 f 0.02 mu Pen G, respectively. The V, and K, values of PGA were estimated from the same plot as 20.84 & 0.03 U ml-’ rnin-’ and
0
0.13 0.12 0.11 -r
0.5MSucmse
L-J
0.14
0.1 0.09
0
l.OMSuCmse
A
o.o~su~mae
0.08 0.07 0.06 0.05 0.04 0.03
0
0.2
0.4
0.6
0.8
1
WI Fig. 7. Lineweaver-Burk plot of PGA from a mutant of E. coli ATCC 11105 in the absence and presence of @5 Mand 1 M sucrose, to determine the K, and V,, values. Equations of best-fit lines, obtained by linear regression and r ialues, are as follows: 0 M sucrose. 1/~=(4.79861+0.23614)X lo-‘+ (1.27984 + @05902) x 10-l 11/S), r= -0.996825; @5 lo-*+(5.70752+ M sucrose, l/v =(6*51061 f0.23948)~ 0.42262)x lo-* (l/S), r= -0.994562; 1 M sucrose, l/v= (7~08070f004693)\x)10-‘~+~(5~56148rtO=10181)~x 1O-2 (l/S), r= - 0.999671.
2.67 f 0.02 ~-IMPen G, respectively, in the absence of sucrose. The comparison of V, and K, values in sucrose-containing PGA to those of sucrose-free PGA in buffer solution ( If,,,, and K,,,J showed that K, and V, values decreased with increasing concentrations of added sucrose, but the percentage of decrease was higher in Km values (Fig. 8). This can be explained by an increase of diffusional resistance to the Pen G in concentrated sucrose media. Results suggest that the more the
Altan Erarsh
138
was higher in sucrose-containing media up to 5 mu concentrations of Pen G (Fig. 7). Consequently the activation energy of sucrose-containing enzyme decreased from 10-l 5 + O-43 to 8.17 k O-58 kcal mol- ‘. This indicates that the presence of sucrose contributes to a decrease in the activation energy of PGA.
loo 80
0
3 6o E
L
40
of
I.,
0
8,
0.2
0.4
I.
I.
0.6
0,8
/
lo
I
SucmwCnnccotmtion.(M)
Fig. 8. Effect of sucrose concentration on V, and K, values of PGA from a mutant of E. coli ATCC 11105 for Pen G hydrolysis.
ACKNOWLEDGEMENTS This work was supported by funds from the NATO-SFS Program (Tu-842 Biotech). The author wishes to kindly acknowledge the contributions of Halil Kocer, a biologist in our Institute, for his technical assistance.
3.6 3.4
REFERENCES
3.2 3 2.8 ' 9
2.6 2.4 2.2 2 1,s
I.6
F., 3
,. 3.05
, , , . , , , , , , , , , , , , .y 3.1
3.15
3.2
3.25
3.3
3.35
[lrr'1*lo".(Kr'
plot for the estimation of activation Fig. 9. Arrhenius energies of PGA from a mutant of E. coli ATCC 11105 in the absence and presence of 1 M sucrose. Equations of bestfit lines obtained by linear regression and r values are as In v=(1~87816f0~0695197)~ lOI+ follows: buffer, (-5.10879f0.217429)~ 10m3 (l/T), r= -@995502; sucrose, In v=(1~56448~0~0914875)x lOl+(-4.10958+ 0.293123) X 10m3 (l/T), r= - 0.989978.
water structure of enzyme was affected by increased sucrose concentration, the more the initial activity of enzyme decreased. Water is a reactant of hydrolytic reactions and, as sucrose tends to make a fraction of the water unavailable, the water activity and thus the enzyme activity are reduced. Effect of sucrose on the activation energy of PGA The activation energy of PGA in the absence and presence of 1 M sucrose was calculated from the slope of the Arrhenius plot (Fig. 9) as 10.15 t-O-43 and 8.17 + 0.58 kcal mol-‘, respectively. In general, the activation energy of a reaction is expected to be increased when a decrease in the reaction rate occurs. Within the temperature ranges tested from 35°C to 70°C the Arrhenius plot of Pen G hydrolysis was close to first order. The initial reaction rate of Pen G hydrolysis
1. Schmid, R. D., Stabilized soluble enzymes. A& Biothem. Eng., 12 (1979) 41-127. of enzymes against 2. Klibanov,- A. M., Stabilization thermal inactivation. Adv. Au.& _. Microbial.. 29 i 1983) l-28. 3. Janecek, s., Strategies for obtaining stable enzymes. Proc. B&hem.. 28 (1993) 435-45. ‘Xu, 2. F., Biton, J. & Thomas, D., 4. Larreta-Garde,‘V., Stability of enzymes in low water activity media. Biocatalysis in Organic Media, ed. J. Tramper & M. D. Lilly Proc. Znt. Symp., Wageningen, The Netherlands, 1986, pp. 247-52. 5. Larreta-Garde, V., Xu, Z. F. & Thomas, D., Behavior of enzymes in the presence of additives. Enzyme Engineerina. ed. H. W. Blanch & A. M. Klibanov. Vol. 9. 1988. ~6’294-8. 6. Erarslan, A., The effect of pH on the kinetics of penicillin G acylase obtained from a mutant of Escherichiu coli ATCC 11105. Proc. Biochem., 28 (1993) 319-24. 7. Erarslan, A. & Kocer, H., Thermal inactivation kinetics of penicillin G acyiase obtained from a mutant derivative of Escherichiu coli ATCC 11105. J. Chem. Technol. Biotechnol., 55 (1992) 79-84. of 8. Erarslan, A. & Giiray, A., Kinetic investigation penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105 immobilized on oxirane-acrylic beads. J. Chem. Technof. Biorechnol., 5 1 ( 1991) 181-95. 9. Erarslan, A., Dagagan, L., Giiray, A., Terzi, 1. & Bermek, E., Several immobilization methods of penicillin acylase purified from Escherichia coli ATCC 11105 and kinetic properties of enzyme immobilized on carboxy methyl cellulose. Doga-Tr. J.. Eng. Env. Sci., 14 (1990) 381-8. 10. Era&m, A., Terzi, I., Giiray, A. & Bermek, E., Purification and kinetics of penicillin G acylase from a mutant strain of Escherichiu coli ATCC 11105. J. Chem. Technol. Biotechnol., Sl(l991) 27-40. 11. Erarslan, A. & Giiray, A., Fermentation of penicillin G acylase by a mutant strain of Escherichia co/i ATCC lllOS.Dojja-Tr.L Biology, 15(1991) 167-74. 12. Erarslan, A., Dagavan, L., Giiray, A., Terzi, i. & Bermek, E., Purification and kinetic properties of penicillin acylase from Escherichia coli ATCC 11105. Doga-Tr. J. Biology, 13 (1989) 8-13.
Themstability
of penicillin G acylase
13. Batchelor, F. R., Chain, E. B., Hardy, T. L., Mansford, K.
R L. & Rolinson, G. N., 6-Aminopenicillanic acid. III. Isolation and purification. Proc. Roy. Sot., B, 154 (1961) 498-508. 14. Shewale, G. J., Kumar, K. K. & Ambekar, G. R., Evaluation and determination of 6-aminopenicillanic acid by pdimethylaminobenzaldehyde. Biotechnol. Techniques, 1 (1987) 69-72.
139
15. Spector, T., Refinement of the Coomassie Blue method of protein quantitation. Anal. Biochem., 86 (1978) 142-6. 16. Sedmak, J. J. & Grossberg, S. E., A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G-250. Anal. Biochem., 79 (1977) 544-52.